Current available biomarkers cannot identify myocardial ischemia without necrosis. To overcome this issue and to increase diagnostic power, we evaluated the activation of the three MAPK pathways, ERK1/2, JNK and p38, in T lymphocytes of patients with acute coronary syndromes (ACS). We included sixty consecutive patients affected by either unstable angina (UA, N = 22), Non- ST-segment elevation MI (NSTEMI, N = 19) or ST-segment elevation MI (STEMI, N = 19). Two separate groups of patients were matched as controls: healthy subjects (CTRL, N = 20) and patients with stable coronary artery disease (CAD, N = 21). MAPK activation in T lymphocytes, measured by phospho-ERK1/2, phospho-JNK and phospho-p38 levels, was assessed by flow cytometry analysis which revealed significantly increased phosphorylated levels of ERK1/2 in patients with UA, compared to controls. In UA patients no significant changes were detected for phospho-JNK compared to both control groups. NSTEMI and STEMI groups showed a statistically significant increase in both phospho-ERK1/2 and phospho-JNK, compared to control groups. All ACS groups demonstrated significantly increased phosphorylation of p38 compared to CTRL, but not CAD. ROC curves showed that a cut-off value of 22.5 intensity of fluorescence for phospho-ERK1/2 was able to significantly discriminate UA patients from patients with stable angina with 78% sensitivity and 90% specificity. Therefore, a differential MAPK activation in T lymphocytes denotes patients with ACS. Indeed, patients with unstable angina are identified with high specificity by activated ERK1/2 and normal JNK levels. These data could represent a valuable new molecular signature to be used as specific biomarkers for the diagnosis of unstable angina within ACS.